PRRSV vaccines

ABSTRACT

The invention relates to the field of PRRS viruses and infectious clones obtained from PRRS viruses. Furthermore, the invention relates to vaccines and diagnostic assays obtainable by using and modifying such infectious clones of PRRS viruses. The invention provides a porcine reproductive and respiratory syndrom virus (PRRSV) replicon having at least some of its original PRRSV nucleic acid deleted, said replicon capable of in vivo RNA replication, said replicon further having been deprived of at least some of its original PRRSV nucleic acid and/or having been supplemented with nucleic acid derived from a heterologous microorganism.

[0001] The invention relates to the field of PRRS viruses and infectiousclones obtained from PRRS viruses. Furthermore, the invention relates tovaccines and diagnostic assays obtainable by using and modifying suchinfectious clones of PRRS viruses.

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) is apositive-strand RNA virus that belongs to the family of arterivirusestogether with equine arteritis virus (EAV), lactatedehydrogenase-elevating virus (LDV) and simian hemorrhagic fever virus(Meulenberg et al., 1993). Recently, the International Committee on theTaxonomy of Viruses has decided to incorporate this family in a neworder of viruses, the Nidovirales, together with the Coronaviridae(genomic length 28 to 30 kb), and Toroviridae (genomic length 26 to 28kb). The order Nidovirales represents enveloped RNA viruses that containa positive-stranded RNA genome and synthesize a 3′ nested set ofsubgenomic RNAs during replication. The subgenomic RNAs of coronavirusesand arteriviruses contain a leader sequence which is derived from the 5′end of the viral genome. The subgenomic RNAs of toroviruses lack aleader sequence. Whereas the ORFs 1a and 1b, encoding the RNA dependentRNA polymerase, are expressed from the genomic RNA, the smaller ORFs atthe 3′ end of the genomes of Nidovirales, encoding structural proteins,are expressed from the subgenomic mRNAs.

[0003] A replicon herein is defined as derived from a recombinantnucleic acid. Although genomic information regarding PRRSV is nowemerging, it is for example not known where deletions or modificationsin the PRRSV genome can be located so that the resulting recombinantnucleic acid can be used as a functional replicon allowing in vivo RNAreplication, be it in (complementary) cells expressing essential (PRRS)viral proteins (such as polymerase or structural (envelope) proteins ornot, or allowing independent in vivo RNA replication in animals, such aspigs, after vaccination with a vaccine comprising a nucleic acidencoding such a PRRS replicon.

[0004] PRRSV (Lelystad virus) was first isolated in 1991 by Wensvoort etal. (1991) and was shown to be the causative agent of a new disease nowknown as porcine reproductive respiratory syndrome (PRRS). The mainsymptoms of the disease are respiratory problems in pigs and abortionsin sows, sometimes complicated by sow-mortality. Although the majoroutbreaks, such as observed at first in the US in 1987 and in Europe in1991, have diminished, this virus, in its various virulent orless-virulent forms, still causes major economic losses in herds in theUS, Europe, and Asia.

[0005] PRRSV preferentially grows in alveolar lung macrophages(Wensvoort et al., 1991). A few cell lines, such as CL2621 and othercell lines cloned from the monkey kidney cell line MA-104 are alsosusceptible to the virus. Some well known PRRSV strains are known underaccession numbers CNCM I-1102, I-1140, I-1387, I-1388, ECACC V93070108,or ATCC VR 2332, VR 2385, VR 2386, VR 2429, VR 2474, and VR 2402. Thegenome of PRRSV is 15 kb in length and contains genes encoding the RNAdependent RNA polymerase (ORF1a and ORF1b) and genes encoding structuralproteins (ORFs 2 to 7; Meulenberg et al., 1993 and Meulenberg et al.,1996). ORF5 encodes the major envelope glycoprotein, designated GP₅. TheORFs 2, 3, and 4 encode glycoproteins designated GP₂, GP₃, and GP₄,respectively. These glycoproteins are less abundantly present inpurified virions of the Lelystad virus isolate of PRRSV. The GP₅ proteinforms a di-sulfide-linked heterodimer with the membrane protein Mencoded by ORF6. The nucleocapsid protein N is encoded by ORF7. Theanalysis of the genome sequence of PRRSV isolates from Europe and NorthAmerica, and their reactivity with monoclonal antibodies has proven thatthey represent two different antigenic types. The isolates from thesecontinents are genetically distinct and must have diverged from a commonancestor relatively long ago (Murtaugh et al., 1995).

[0006] Pigs can be infected by PRRSV via the oronasal route. Virus inthe lungs is taken up by lung alveolar macrophages and in these cellsreplication of PRRSV is completed within 9 hours. PRRSV travels from thelungs to the lung lymphnodes within 12 hours and to peripherallymphnodes, bone marrow and spleen within 3 days. At these sites, only afew cells stain positive for viral antigen. The virus is present in theblood during at least 21 days and often much longer. After 7 daysantibodies to PRRSV are found in the blood. The combined presence ofvirus and antibody in PRRS infected pigs shows that the virus infectioncan persist for a long time, albeit at a low level, despite the presenceof antibody. During at least 7 weeks the population of alveolar cells inthe lungs is different from normal SPF lungs.

[0007] PRRSV needs its envelope to infect pigs via the oronasal routeand the normal immune response of the pig thus entails among others theproduction of neutralising antibodies directed against one or more ofthe envelope proteins; such antibodies can render the virusnon-infective. However, once in the alveolar macrophage, the virus alsoproduces naked capsids, constructed of RNA encapsidated by the M and/orN protein, sometimes partly containing any one of the glycoproteins. Theintra- and extracellular presence of these incomplete viral particles or(partly) naked capsids can be demonstrated by electron microscopy.Sometimes, naked capsids without a nucleic acid content can be found.The naked capsids are distributed through the body by the bloodstreamand are taken up from the blood by macrophages in spleen, lymphnodes andbonemarrow. These naked but infectious viral capsids can not beneutralised by the antibodies generated by the pig and thus explain thepersistence of the viral infection in the presence of antibody. In thisway, the macrophage progeny from infected bonemarrow cells is spreadingthe virus infection to new sites of the body. Because not all bonemarrowmacrophage-lineage cells are infected, only a small number ofmacrophages at peripheral sites are infected and produce virus. PRRSVcapsids, consisting of ORF7 proteins only, can be formed in the absenceof other viral proteins, by for instance infection of macrophages with arecombinant pseudorabies-ORF7 vector virus. The PRV virus wasmanipulated to contain ORF7 genetic information of PRRSV. After 18 hourspost infection, the cytoplasm of infected cells contains large numbersof small, empty spherical structures with the size of PRRS virusnucleocapsids.

[0008] Although live-attenuated and killed PRRSV vaccines are nowavailable, it has been shown that in general these are not immunogenicenough or are too virulent for specific groups of pigs, i.e. for youngpiglets or sows in the third trimester of pregnancy. It is clear that aPRRSV vaccine that is not sufficiently immunogenic will not stand up inthe market. However, several of the existing immunogenic vaccines arenot safe illustrating the need for attenuated PRRSV vaccines withreduced virulence.

[0009] Furthermore, again under specific circumstances, several of theexisting vaccines spread within a population, and may inadvertentlyinfect other pigs that need not or should not be vaccinated,illustrating the need for non-spreading PRRSV vaccines.

[0010] Furthermore, the existing vaccines can in general not bedistinguished from wild type field virus, illustrating the need for aso-called marker vaccine, obtained for example by mutagenesis of thegenome, so that vaccinated pigs can be distinghuished from fieldvirus-infected pigs on the basis of differences in serum antibodies.

[0011] In addition, PRRS vaccines, being so widely used throughout theworld, and being in general not infectious to other animals but pigs,would be attractive candidate vaccines to carry foreign antigens derivedfrom other (porcine) pathogens to provide for protection against thoseother pathogens, illustrating the need for PRRSV carrier or vectorvaccines allowing vaccination against those other pathogens or allowingpositive marker identification.

[0012] It goes without saying, that PRRSV vaccines combining one or moreof these features would be preferred. It is an object of the presentinvention to provide solutions to these needs.

[0013] The invention provides a porcine reproductive and respiratorysyndrome virus (PRRSV) replicon having at least some of its originalPRRSV nucleic acid deletions, herein also comprising substitutions, saidreplicon capable of in vivo RNA replication, said replicon furtherhaving been deprived of at least some of its original PRRSV nucleic acidand/or having been supplemented with nucleic acid derived from aheterologous micro-organism.

[0014] Surprisingly, it has been found that the genome of PRRSV can bedeprived of quite a large amount of its nucleic acid. An independent andfunctional PRRSV replicon capable of independent in vivo RNA replicationcan still exist if the stretch, or fragments thereof, of nucleic acidencoding the ORF2-ORF6, but not an essential element from the ORF7protein, is deleted and/or modified. Having a replicon wherein such alarge stretch of nucleic acid has been deleted or modified opens up alarge capacity for the addition to said replicon of heterologous nucleicacid from any other organism than PRRSV, thereby providing a PRRSVvector replicon with large carrier capacities. Herewith, the inventorprovides identification of specific nucleic acid regions in the genomeof porcine reproductive and respiratory syndrome virus, that areimportant for attenuation of the virus, for making it non- or littlespreading or for the introduction of a marker, without crippling theviral nucleic acid so much that it can no longer provide in vivo RNAreplication. Furthermore, the inventor demonstrates that a PRRSVreplicon can be used as vector for the expression of foreign antigens,preferably derived from other (porcine) pathogens, allowing vaccinationagainst those other pathogens and allowing positive markeridentification. The minimal sequence requirements for a PRRSV repliconor PRRSV vector replicon as provided by the invention are essentialelements comprising the 5′ noncoding region-ORF1a-ORF1b-ORF7-3′noncoding region, (e.g. from the PRRSV polymerase region) whereby theORF7 coding region can be deleted further for example according to thedata shown in FIG. 2. In a preferred embodiment, the invention providesa PRRSV replicon or vector at least comprising essential elements fromthe PRRSV polymerase region for example as described below and/orcomprising at least nucleic acid derived from a essential region of 44nucleotides between nucleotides 14642 to 14686 in the ORF7 gene (asidentified in the nucleic acid sequence of the Lelystad virus isolate ofPRRSV, however, the skilled person can easily determine by alignmentwherein in any other PRRSV genome said essential element is located).

[0015] In another preferred embodiment, the invention provides a PRRSVreplicon comprising at least nucleic acid derived from essentialsequence elements from ORF1a and ORF1b, or from the PRRSV polymeraseregion and having nucleic acid from ORF2, ORF 3, ORF 4, ORF 5, ORF 6, ornon-essential elements from ORF7 deleted, allowing insertion of foreignnucleic acid, thereby providing a PRRSV vector replicon having foreignantigen coding capacities. This in contrast to WO98/55626 where thehomologous polymerase is replaced with a heterologous Arteriviral one toexpress ORF2-ORF7, essentially without disclosing expression of foreignantigens derived from other (porcine) pathogens to provide forprotection against those other pathogens allowing vaccination againstthose other pathogens (let alone wherein the PRRSV genome nucleic acidencoding foreign antigens may be located for providing a PRRSV vectorreplicon or which essential sequence elements should remain).

[0016] The replicase polyprotein of PRRSV encoded by ORF1 is thought tobe cleaved in 13 processing end-products (designated nonstructuralproteins—nsps) and a large number of intermediates. The polyprotein iscleaved by protease domains located in nsp1α, nsp1β, nsp2 and nsp4.Essential PRRSV RNA-dependent RNA polymerase and nucleosidetriphosphate-binding/RNA Helicase motifs were identified in nsp9 andnsp10, respectively. Another conserved (essential) domain was found innsp11, a conserved Cys/His-rich domain was found in nsp10. It has forexample been shown that the latter protein plays a role in subgenomicmRNA synthesis.

[0017] In a further embodiment, the invention provides a PRRSV repliconcapable of independent in vivo RNA replication wherein said replicon isa RNA transcript of an infectious copy cDNA. It has been shown for manypositive strand RNA viruses that their 5′ and/or 3′ noncoding regionscontain essential signals that control the initiation of plus- andminus-strand RNA synthesis. It was not determined for PRRSV whetherthese sequences alone are sufficient for replication. As for most RNAviruses, PRRSV contains a concise genome and most of the geneticinformation is expected to be essential. Furthermore, the maximumcapacity for the integration of foreign genes into the PRRSV genome isnot yet known. An extra limitation is that the ORFs encoding thestructural proteins of PRRSV are partially overlapping. The introductionof mutations in these overlapping regions often results in two mutantstructural proteins and therefore is more often expected to produce anonviable virus.

[0018] The production of an infectious clone allowed us to analysereplication signals in the genome of PRRSV. In this study we have mappedcis-acting sequence elements required for replication by introducingdeletions in the infectious clone. Surprisingly, we have shown that alsocis-acting sequence elements from the region of the genome encodingstructural proteins are essential for proper replication. We have shownthat transcripts derived from cDNA clones lacking the ORF7 gene are notreplicated. A more systematic deletion analysis showed that a region of44 nucleotides between nucleotides 14642 to 14686 in the ORF7 gene wasessential for replication of RNA of PRRS. This was an interestingfinding, since the sequences essential for replication of most positivestrand RNA viruses are present in the 5′ and 3′ noncoding regions. It isan important finding for studies who's aim is to develop viral repliconswhich can only be rescued in complementing cell lines expressing thedeleted ORFs. The minimal sequence requirements for these RNAs arelocated in the 5′ noncoding region-ORF1a-ORF1b-ORF7-3′ noncoding region.Viral RNA's or replicons containing these sequence elements supplementedwith a selection of fragments from other PRRSV open reading frames orfragments of open reading frames expressing antigens of other(heterologous) pathogens can be packaged into virus particles when theproteins essential for virus assembly are supplied in trans. When theseparticles are given to pigs, for example as vaccine, they will enterspecific host cells such as macrophages and virus- or heterologousantigens are expressed and induce immune responses because of thereplicating RNA. However, since the RNA does not express (all) theproteins required for packaging and the production of new particles, thereplicon can not spread further, creating an extremely efficient, butsafe and not-spreading recombinant vaccine effective against PRRSVand/or heterologous pathogens.

[0019] In a preferred embodiment, the invention provides a repliconaccording to the invention incapable of N-protein capsid formation. Forexample, two Cys residues are present at positions 27 and 76 in the Nprotein sequence and mutating or deleting Cys-27 and Cys-76 from the Nprotein inhibits the production of infectious particles of PRRSV. TheORF7 gene encoding the N protein was mutated as such that the Cysresidues were substituted for Asn and Leu residues, respectively,however, substitution with another amino acid, or deletion of the codingsequence, leads to the desired result as well, as for example can beseen below.

[0020] The Cys-27 and Cys-76 mutations were subsequently introduced inthe infectious clone pABV437 of the Lelystad virus isolate of PRRSV,resulting in plasmids pABV534-536 (Cys-27→Asn) and pABV472-475(Cys-76→Leu). RNA was transcribed from these mutated infectious clonesand transfected to BHK-21 cells. The structural proteins were properlyexpressed, these mutant RNAs were replicated and subgenomic RNAssynthesized. However, infectious particles were not secreted, since thetransfer of the supernatant of the transfected BHK-21 cells tomacrophages did not result in the production of viral proteins in themacrophages nor in the induction of a cpe.

[0021] Thus, these residues are essential for a proper structure orfunction or both of the N protein in virus assembly of PRRSV. The Nprotein is involved in the first steps in virus assembly, the binding ofthe viral genomic RNA and formation of the capsid structure. Sincetranscripts of genomic length cDNA clones containing the Cys-27 and/orCys-76 deletion replicated at the wild type level, the mutations in theCys residues destroy the binding of the RNA by the N protein.Alternatively, they induce a different structure of the N protein thatinhibits the formation of proper capsids. The defect in theencapsidation of the viral RNA genome can be complemented by wild type Nprotein transiently expressed or continuously expressed in a (BHK-21)cell line. In this way a virus is produced that is able to complete onlyone round of infection/replication. Therefore such a virus is consideredto be a very safe vaccine for protection against PRRSV in pigs.

[0022] In another example, the invention provides a replicon incapableof N-protein capsid formation wherein substitutions in the genomeencoding the N protein area containing two antigenic regions designatedB and D inhibited the production of infectious virus particles. The Bregion comprises amino acids 25-30 (QLCQLL), D region; amino acids 51-67(PEKPHFPLAAEDDIRHH) and amino acids 80-90 (ISTAFNQGAGT) of the N proteinof PRRSV. The corresponding sites in VR2332 and other American strainsare found when the N proteins of these strains are aligned. Since RNAreplication and subgenomic mRNA synthesis appeared to be at the wildtype level, these mutations most likely prevented the formation ofproper capsids by the N protein.

[0023] The invention furthermore provides a replicon according to theinvention wherein a marker allowing serological discrimination has beenintroduced. For example, mutagenesis of a single amino acid in the Dregion (Asp-62 or a.a. corresponding thereto) of protein N results in areplicon that has a different MAb binding profile from PRRSV and allother PRRSV viruses. Such a replicon induces a different spectrum ofantibodies in pigs, compared to these other PRRSV isolates. Therefore itcan be differentiated from field virus on the basis of serum antibodiesand is an excellent mutant for further development of marker vaccinesagainst PRRSV.

[0024] The above example involves a subtle modification resulting in areplicon useful for a marker vaccine. However, more extensive changesare now also possible, knowing that it is allowed to partly or fullydelete the nucleic acid encoding the structural proteins 2, 3, 4, 5,and/or 6 without tampering with the replicative properties of theresulting replicon. A PRRSV replicon lacking one or more (antigenic)fragments of these structural proteins has the advantage that no immunerespons, more specifically no antibodies, against these deletedfragments will be formed, for example after vaccination with a vaccinecomprising such a replicon. Again, such a replicon induces a differentspectrum of antibodies in pigs, compared to wild type PRRSV. Thereforeit can be differentiated from field virus on the basis of serumantibodies and is an excellent mutant for further development of markervaccines against PRRSV.

[0025] Furthermore, the invention provides a replicon comprising anucleic acid modification in a virulence marker of PRRSV. Virulencemarkers of PRRSV have not been elucidated, despite the fact that variousdifferences in virulence have been observed. However, for successfullyattenuating a PRRSV or replicon thereof, such knowledge helps inselecting the least virulent, but most immunogenic replicon or viruspossible. Now that it is known that deleting or modifying the ORF2 toORF 6 region is possible without effecting the in vivo RNA replicativeproperties, such virulence markers can easily be detected. For example,the invention provides replicon comprising a nucleic acid modificationin ORF 6 encoding the membrane spanning M-protein. It has been foundthat the membrane protein is influencing the virus assembly, thestability of the virus, or the virus entry in macrophages, all factorscontributing to the virulence of PRRSV. The M protein is the mostconserved structural protein among arteriviruses and coronaviruses. Theprotein is an integral membrane protein containing three N-terminalhydrophobic membrane spanning domains (Rottier, 1995). The protein spansthe membrane three times leaving a short N-terminal domain outside thevirion and a short C-terminal domain inside the virion. The M protein ofcoronaviruses was shown to play an important role in virus assembly(Vennema et al., 1996), but was then not determined to be a virulencefactor. In particular, the invention provides a replicon wherein saidmodification modifies protein M in between its second and third membranespanning fragment, essential in determining virulence of a specificPRRSV isolate. For example, the invention provides a replicon comprisingvABV575. A Thr-59→Asn mutation is located between the second and thirdmembrane spanning fragment of M in vABV575. This mutation influencesvirus assembly, the stability of the virus, or virus entry in the PAMs.

[0026] The invention furthermore provides a replicon according to theinvention wherein said heterologous micro-organism comprises a pathogen.Since PRRSV specifically infects macrophages, it can be used as a vectorfor the delivery of important antigens of other (respiratory) agents tothis specific cell of the immune system. The infectious cDNA cloneenables us to introduce site specific mutations, deletions andinsertions into the viral genome.

[0027] In a preferred embodiment, the invention provides a repliconwherein said pathogen is a virus. We have successfully used PRRSV as avector for the expression of a foreign protein anigen, an HA epitope ofthe haemagglutinin of influenza A virus. Recombinant PRRSV vectorreplicons were engineered that produced the HA tag fused to the N- orC-terminus of the N protein. In addition, an PRRSV mutant was createdthat contained the HA-tag as well as the protease 2A offoot-and-mouth-disease virus (FMDV) fused to the N terminus of the Nprotein.

[0028] Furthermore, the invention provides a vaccine comprising areplicon or vector replicon according to the invention. PRRSV vaccinesare now provided with specified antigenicity or immunogenicity that arein for example in addition safe enough for specific groups of pigs, i.e.for young piglets or sows in the third trimester of pregnancy.

[0029] Furthermore, the invention provides non-spreading PRRSV vaccines,comprising a replicon or vector replicon for example incapable ofN-protein capsid formation, or incapable of further infection due to theabsence of (fragments of) structural proteins encoded by ORF 2 to 6,without hampering its in vivo RNA replication properties, therebyallowing the production of proteins against which an immune response isdesired.

[0030] Furthermore, the invention provides a vaccine that can bedistinguished from wild type field virus, a so-called marker vaccine,obtained for example by mutagenesis of the genome, so that vaccinatedpigs can be distinguished from field virus-infected pigs on the basis ofdifferences in serum antibodies.

[0031] In addition, PRRS vaccines, being so widely used throughout theworld, and being in general not infectious to other animals but pigs,are now provided as vector vaccines to carry foreign antigens derivedfrom other (porcine) pathogens, allowing vaccination against those otherpathogens and allowing positive marker identification.

[0032] Use of a vaccine according to the invention is especially usefulfor vaccinating pigs, sine the PRRSV is in general very host specificand replicates in macrophages of pigs, thereby targeting an importantantigen presenting cell of the immune system.

[0033] The invention is further explained in the detailed description,without limiting the invention.

DETAILED DESCRIPTION

[0034] 1. Mutation of Cys-27 and Cys-76 in the N Protein Inhibits theProduction of Infectious Particles of PRRSV

[0035] The nucleocapsid protein N (expressed by ORF7) is present as amonomer in purified virions of PRRSV. However, in some experiments wealso detected a homodimer of N. For instance when the N protein wasimmunoprecipitated from purified virions with N-specific MAbs andelectrophorezed on a sodium dodecyl sulfate polyacrylamide gel(SDS-PAGE), a protein of 15 kDa was predominantly observed under reducedconditions, whereas a homodimer of 30 kDa was predominantly observedunder nonreduced conditions (Meulenberg et al., 1996). However, whencompounds such as N-methyl maleimide or iodoacetamide were used toprevent the formation of nonspecific disulfide bonds, these dimers of Nwere not detected. This indicated that dimers of N are formed due to theformation of nonspecific disulfide bonds during the processing of celllysates for analysis. Two cystein residues are present in the N proteinsequence. The question raised which of the cysteine residues wasresponsible for the formation of nonspecific disulfide bonds and whetherthe cysteine residues are important for the structure and function ofthe N protein. To answer this question we mutated the two cysteinresidues individually in the infectious cDNA clone of PRRSV and studiedthe infectivity of the resulting mutant viral genomic RNAs.

[0036] 2. Introduction of a Marker in the N Protein

[0037] The N protein of PRRSV contains 4 antigenic sites, designated A-D(Meulenberg et al., 1998). Two sites, B and D, contain epitopes that areconserved in European and North American isolates of PRRSV. To produceviruses that can be serologically distinguished from wild type viruses,mutations in the B and D domain that disrupt the binding of N-specificMAbs were introduced in the infectious cDNA clone of PRRSV. Transcriptsof the resulting mutant full length cDNA clones were analyzed for RNAreplication by detecting the expression of structural proteins andproduction of infectious virus.

[0038] 3. Elucidation of Replication Signals Present in the RegionEncoding Structural Proteins of Lelystad Virus

[0039] Positive strand RNA viruses contain 5′ and 3′ noncoding regionswhich are essential for replication. The RNA sequences at the 5′ and 3′end usually have a specific secondary structure which is recognized bythe viral RNA dependent RNA polymerase to initiate positive and negativestrand synthesis and in the case of arteriviruses subgenomic RNAsynthesis. We deleted the ORF7 gene from the infectious clone of PRRSV(Meulenberg et al., 1998) in a first attempt to generate a defective RNAreplicon that could be complemented for production of infectiousparticles, when transfected to a cell expressing the N protein. The ORF7gene was precisely deleted, without affecting the 3′ noncoding region ofthe virus. Surprisingly, the RNA of this deletion mutant did notreplicate in BHK-21 cells. This suggested that RNA replication signalsare present in the coding region of ORF7. The purpose of this study wasto further localize these replication signals. By expensive deletionanalysis of the coding region and upstream sequences of ORF7 we wereable to identify a region of 44 nucleotides in the ORF7 gene that isimportant for replication of RNA of PRRSV.

[0040] 4. Production of an Attenuated PRRSV Virus by Deletion of theNdeI Site in ORF6.

[0041] Recently, we have established an infectious clone cDNA clone ofPRRSV (Meulenberg et al., 1998). The full length cDNA clone contains twoNdeI sites, the first at nucleotide 12559 (ORF3) and the second atnucleotide 14265 (in ORF6) in the genome sequence. To facilitatemutagenesis and exchange of fragments in the region encoding thestructural proteins (ORFs 2 to 7) of the virus, we destroyed the secondNdeI site by PCR-directed mutagenesis. This resulted in an amino acidsubstitution at position 59 in the M protein (Thr→Asn). The growthproperties of the virus produced from the mutated full length cDNA clonecontaining a unique NdeI site was analysed.

[0042] 5. Lelystad Virus as a Vector for the Expression of ForeignAntigens or Proteins.

[0043] The generation of an infectious cDNA clone of PRRSV (Meulenberget al., 1998) is a major breakthrough in PRRSV research and opens up newpossibilities for the development of new viral vectors. Since PRRSVspecifically infects macrophages, it can be used as a vector for thedelivery of important antigens of other (respiratory) agents to thisspecific cell of the immune system. The infectious cDNA clone enables usto introduce site specific mutations, deletions and insertions into theviral genome. However, it is still not known which regions of the PRRSVgenome are essential or allow mutagenesis. As for most RNA viruses,PRRSV contains a concise genome and most of the genetic information isexpected to be essential. Furthermore, the maximum capacity for theintegration of foreign genes into the PRRSV genome is not yet known. Anextra limitation is that the ORFs encoding the structural proteins ofPRRSV are partially overlapping. The introduction of mutations in theseoverlapping regions results in two mutant structural proteins andtherefore is more often expected to produce a nonviable virus.

[0044] The aim of this study was to identify regions in the PRRSV genomethat allow the introduction of foreign antigens that will be exposed tothe immune system of the pig after infection with the mutant virus. In afirst approach we have selected a small epitope of 9 amino acids ofhuman haemagglutinin of influenza A for expression in PRRSV.

[0045] METHODS

[0046] Cells and Viruses

[0047] BHK-21 cells were grown in BHK-21 medium (Gibco BRL), completedwith 5% FBS, 10% tryptose phosphate broth (Gibco BRL), 20 mM Hepes pH7.4 (Gibco BRL) and 200 mM glutamine, 100 U/ml penicillin and 100 μg/mlstreptomycin. Porcine alveolar lung macrophages (PAMs) were maintainedin MCA-RPMI-1640 medium, containing 10% FBS, 100 μg/ml kanamycin, 200U/ml penicillin and 200 μg/ml streptomycin. Virus stocks were producedby serial passage of recombinant PRRSV viruses secreted in the culturesupernatant of tranfected BHK-21 cells on PAMs. Virus was harvested whenPAMs displayed cytopathic effect (cpe) usually 48 hours after infection.Virus titers (expressed as 50% tissue culture infective doses [TCID₅₀]per ml) were determined on PAMs using end point dilution (Wensvoort etal., 1986).

[0048] Mutagenesis

[0049] 1. Mutagenesis of Cys-27 and Cys-76.

[0050] The Cys-27 was mutated to Asn by PCR-directed mutagenesis withprimers LV108 and LV97. The sequences of primers used in this study arelisted in Table 1 . The generated PCR fragment was digested with HpaIand PflmI and inserted in the ORF7 gene in pABV431 digested with thesame enzymes. This resulted in plasmid pABV451 The Cys-76 was mutated toLeu by PCR-directed mutagenesis with primers LV108 and LV100. Thegenerated fragment was digested with HpaI and ClaI and inserted in theORF7 gene in pABV431 digested with the same enzymes. This resulted inpABV452. The mutated ORF7 genes were subsequently transferred to thegenomic-length cDNA clone pABV437(Meulenberg et al., 1998) with theunique HpaI (nt 14581) and PacI (nt 14981) site, to create plasmidspABV534-536 (Cys-27→Asn) and plasmids pABV472-475 (Cys-76→Leu; FIG. 1).

[0051] 2. Mutagenesis of Antigenic Site B and D in the N Protein

[0052] Antigenic sites B (amino acids 25-30) and D (amino acids 51-67and 80-90) of the N protein of PRRSV were mutated by substitution of theamino acids in this region for the corresponding amino acids ofrespectively EAV and LDV. Plasmids pABV455, pABV463, and pABV453containing these respective mutation were described previously inMeulenberg et al. (1998). In addition, the Asp at position 62 in the Dregion of the N protein was mutated to a Tyr in a PCR with primers LV108and LV188. The sequences of these primers are shown in Table 1. The PCRfragment was digested with HpaI and ClaI and inserted in the ORF7 genein pABV431 digested with the same enzymes. This resulted in pABV582. TheORF7 genes containing the mutations were inserted in pABV437 using theunique HpaI (nt 14581) and PacI (nt 14981) (FIG. 1).

[0053] 3: Creation of Deletion Mutants in the Full-Length cDNA Clone ofPRRSV

[0054] Several deletions were made in the full-length cDNA clone ofpABV437 of PRRSV (FIG. 2). First, ORF2, ORF3, ORF4, ORF5 and the 5′ halfof ORF6 were deleted. pABV437 was digested with EcoRI and NheI and thesites were made blunt with Klenow fragment (Pharmacia Biotech). Thefragment was purified and ligated. This resulted in clone plasmidpABV594. Second, ORF7 was deleted from the infectious copy of PRRSV. Forthis purpose, the infectious full-length cDNA clone pABV442 thatcontains a SwaI restriction site directly downstream of the stopcodon ofORF7, was digested with HpaI and SwaI and ligated. This resulted inclone plasmid pABV521. Third, to delete the 3′ end of ORF6,PCR-mutagenesis was performed with primers LV198 and LV199. The primersused in PCR-mutagenesis are listed and described in Table 1. Thegenerated product was digested with HpaI and NheI and ligated in thecorresponding sites of pABV437. This resulted in plasmid pABV627.Fourth, several deletions in and upstream of the coding region of ORF7were made. PCR-mutagenesis was performed with forward primers LV188-191or LV195-197 and reversed primer LV112. The generated products weredigested with HpaI and PacI and ligated in the same restriction sites ofpABV437, resulting in plasmids pABV602-605 and pABV625-627. Plasmidswere transformed to Escherichia coli DH5α and grown at 32° C. and 20 μgkanamycin per ml. For each construct two clones containing fragments oftwo independent PCRs were sequenced to confirm the correct sequence ofthe clones. The resultant mutants are shown in FIG. 2.

[0055] 4. Mutagenesis of the NdeI Site at Position 14265 in theInfectious cDNA Clone pABV437 of PRRSV

[0056] To mutate the NdeI site at position 14265 a fragment of 1.7 kbwas amplified by PCR using primers LV27 (nt 12526) and LV182 (nt 14257;Table 1) Primer LV182 contains an AseI site. AseI and NdeI havecompatible ends, but ligation of their ends to each other destroys bothrestriction sites. The PCR fragment was digested with NdeI and AseI andligated in pABV437 digested with NdeI. The full length clone pABV575(FIG. 3) that contained the PCR fragment in the proper orientation,lacked the NdeI site at position 14265 and had no other mutationsbetween 12559 and 14265 due to PCR errors was selected for furtheranalysis.

[0057] 5: Construction of Full-Length Genomic cDNA Mutants of PRRSVEncoding an Antigenic HA tag

[0058] PCR-mutagenesis was used to create mutants in the infectiousclone of PRRSV. First, a sequence of 27 nucleotides encoding an epitopeof the human haemagglutinin of influenza A (HA-tag; Kolodziej et al.,1991) was introduced directly downstream of the start codon of ORF7 inthe PacI mutant of the genome-length cDNA clone of Lelystad Virus(pABV437; Meulenberg et al., 1998). Two sequential PCRs were performedwith primers LV192 and LV112 and with primers LV193 and LV112. Primersused to create the PCR-fragments are listed and described in Table 1.Second, both this HA-tag and a sequence of 51 nucleotides encoding theprotease 2A of FMDV (Percy et al., 1994) were introduced directlydownstream of the startcodon of ORF7. Two sequential PCR-reactions wereperformed with primers LV139 and LV112 and with LV140 and LV112. Third,the HA-tag was introduced at the 31 end of the ORF7 gene in a PCR withprimers LV108 and LV194. The three PCR fragments obtained were digestedwith HpaI and PacI and ligated into pABV437 digested with the sameenzymes. Standard cloning procedures were performed essentially asdescribed in Sambrook et al., (1989). Plasmids were transformed intoEscherichia coli DH5α and grown at 32° C. and 20 μg kanamycin per ml.For each construct two clones containing fragments of two independentPCRs were sequenced to confirm the correct sequence of the clones.Introduction of the HA epitope at the 5′ end of ORF7 resulted in thegeneration of clone pABV525, introduction of both the HA-tag and theprotease 2A at the 5′ end of ORF7 resulted in clone pABV523, and theintroduction of the HA-epitope at the 3′ end of ORF7 resulted in clonepABV526 (FIG. 4).

[0059] Sequence Analysis

[0060] The generated cDNA clones were analyzed by oligonucleotidesequencing. Oligonucleotide sequences were determined with the PRISMReady Dye Deoxy Terminator cycle sequencing kit and the automaticsequencer (Applied Biosystems).

[0061] In Vitro Transcription and Transfection of RNA

[0062] Full-length genomic cDNA clones and derivatives thereof werelinearized with PvuI, which is located directly downstream of thepoly(A) stretch. The linearized plasmids were precipitated with ethanoland 1.5 μg of these plasmids was used for in vitro transcription with T7RNA polymerase by the methods described for SFV by Liljeström and Garoff(1991). The in vitro transcribed RNA was precipitated with isopropanol,washed with 70% ethanol and stored at −20° C. until use.

[0063] BHK-21 cells were seeded in 35-mm wells (approximately 10⁶cells/well) and were transfected with 2.5 μg in vitro transcribed RNAmixed with 10 ml lipofectin in optimem as described earlier (Meulenberget al., 1998). Alternatively, RNA was introduced in BHK-21 cells in20-mm wells with 0.5 μg in vitro transcribed RNA mixed with 2 mllipofectin in optimem. The medium was harvested 24 h after transfection,and transferred to CL2621 cells or PAMs to rescue infectious virus.Transfected and infected cells were tested for expression of PRRSVproteins by an immunoperoxidase monolayer assay (IPMA), essentially asdescribed by Wensvoort et al. (1986). Monoclonal antibodies (MAbs)122.14, 122.1, and 126.3 directed against respectively the GP₃, GP₄, Mprotein (van Nieuwstadt et al., 1996) were used for staining in thisassay. A panel of MAbs (122.17, 125.1, 126.9, 126.15, 130.2, 130.4,131.7, 131.9, 138.22, WBE1, WBE4, WBE5, WBE6, SDOW17, NS95, and NS99)directed to four different antigenic sites A-D were used to study theexpression of the N protein (Meulenberg et al., 1998). MAb 12CAS wasused to detect the expression of the HA-epitope and was purchased fromBoehringer Mannheim. In addition, we analyzed the expression of PRRSVproteins by metabolic labeling of transfected or infected cells,followed by immunoprecipitation using specific monoclonal antibodies orpeptide sera directed to the structural proteins of PRRSV, as describedby Meulenberg et al (1996).

[0064] Sequence Analysis of Genomic RNA of Recombinant Viruses

[0065] The culture supernatant of the PAMs infected with passage 3 ofthe HA-expressing viruses was used to analyze viral RNA by RT-PCR. Avolume of 500 μl proteinase K buffer (100 mM Tris-HCl [pH 7.2], 25 mMEDTA, 300 mM NaCl, 2% [wt/vol] sodium dodecyl sulfate) and 0.2 mgProteinase K was added to 500 μl supernatant. After incubation for 30minutes at 37° C., the RNA was extracted with phenol-chloroform andprecipitated with ethanol. The RNA was reverse transcibed with primerLV76. Then, PCR was performed with primers LV37 and LV112 to amplifyfragments of vABV523 and vABV525 and with primers LV37 and LV75 toamplify fragments of vABV526 (Table 1). Sequence analysis was performedto determine whether the mutant viruses at passage 4 still contained theinserted foreign sequences.

[0066] Results

[0067] 1. Mutation of Cys-27 and Cys-76 in full length cDNA clonepABV437 Two Cys residues are present at positions 27 and 76 in the Nprotein sequence. The ORF7 gene encoding the N protein was mutated assuch that the Cys residues were substituted for Asn and Leu residues,respectively. The Cys-27 and Cys-76 mutations were subsequentlyintroduced in the infectious clone pABV437 of the Lelystad virus isolateof PRRSV, resulting in plasmids pABV534-536 (Cys-27→Asn) and pABV472-475(Cys-76→Leu; FIG. 1). RNA was transcribed from these mutated infectiousclones and transfected to BHK-21 cells. These cells stained positivewith N-specific MAbs in IPMA. Analysis of the N protein synthesized bypABV534-536 and pABV472-475 in immuno precipitation and SDS-PAGEindicated that its apparent molecular weight was similar to the wildtype N protein and migrated at 15 kDa under reducing conditions. Next weanalyzed the N protein under nonreducing conditions in the absence ofN-methyl maleimide or iodoacetamide. Under these conditions, the Nprotein expressed by pABV472-475 (Cys-76→Leu) resembled the wild type Nprotein and was mainly detected as a dimer, whereas the N proteinexpressed by pABV534-536 (Cys-27→Asn) was detected as a monomer. Thisindicated that the Cys residue at position 27 was responsible for theformation of nonspecific disulfide bonds. The production of otherstructural proteins such as GP₃, GP₄, and M was also detected in IPMAand immuno precipitation after transfection of full length RNA fromplasmids pABV534-536 (Cys-27→Asn) and pABV472-475 (Cys-76→Leu: FIG. 1).Since the structural proteins were properly expressed, these mutant RNAswere replicated and subgenomic RNAs synthesized. However, infectiousparticles were not secreted, since the transfer of the supernatant ofthe transfected BHK-21 cells to PAMs did not result in the production ofviral proteins in the PAMs nor in the induction of a cpe. Therefore bothCys residues are essential for a proper structure or function or both ofthe N protein in virus assembly.

[0068] 2. Characterization of Full Length cDNA Clones containingMutations in Antigenic Sites of the N Protein of PRRSV

[0069] Site B (amino acids 25-30) and D (amino acids 51-67 and 80-90)are two antigenic regions that are conserved in European and NorthAmerican PRRSV isolates. When we mutated site B and D by substitutingtheir amino acid sequence for the corresponding amino acids of the LDVor EAV N protein, the binding of the N protein by respectivelyB-specific and D-specfic MAbs was disrupted (Meulenberg et al., 1998).To produce a PRRSV virus that is antigenically different from PRRSVfield viruses, we introduced the ORF7 genes containing a mutated B or Dregion in our infectious clone pABV437. This resulted in pABVS27-533,containing a mutated B site (amino acids 25-30), pABV537-539 containinga mutated D domain (amino acids 51-67), and pABV512-515 containing amutated D domain (amino acids 80-90) (FIG. 1). When RNA of these fulllength clones was transfected to BHK-21 cells, these cells stainedpositive with N-specific MAbs at 24 h after transfection. As expected,the N protein expressed by pABV527-533 was recognized by A-, C-, andD-specific MAbs, but not by B-specific MAbs. On the other hand the Nprotein expressed by pABV537-539 and pABV512-515 was recognized by A-,B-, and C-specific MAbs but not by D-specific MAbs. The staining ofcells transfected with the RNA derived from pABVS27-533 , pABV537-539and pABV512-515 with MAbs directed against GP₃, GP₄, and M, was similarto that observed in transfections with RNA derived from wild typepABV437. This suggested that RNA replication and subgenomic mRNAsynthesis were not affected by the mutations. When the supernatant ofthe cells transfected with RNA derived from pABV527-533, pABV537-539 andpABV512-515 was transferred to PAMs, cpe was not produced. Most likely,the mutations in the B and D region destroyed the function of the Nprotein in the formation of a proper capsid structure.

[0070] Since the mutation of 4 amino acids in domain B and 5 or 9 aminoacids in domain D did not allow the generation of infectious particleswe then created a more subtle mutation of 1 amino acid in the D region.We introduced an Asp-62 to Tyr mutation in the N-protein in theinfectious clone of PRRSV. The amino acid Asp-62 in the PRRSV N proteinwas mutated to Tyr by PCR directed mutagenesis and transferred topABV437, resulting in pABV600. RNA transcribed from pABV600 wastranfected to BHK-21 cells. These cells stained positive with MAbsdirected against GP₃, GP₄, M and N. At 24 h after transfection,suggesting that the RNA was replicated and subgenomic mRNAs weresynthesized. When the supernatant of the BHK-21 cells transfected withtranscripts from pABV600 was transferred to PAMs, cpe was detected at2-3 days after inoculation. The infected cells stained positive withPRRSV specific MAbs, which further confirmed that infectious virus wasproduced. Therefore, the mutation of Asp-62 to Tyr in the N protein istolerated in the virus and does not destroy the function of the Nprotein. The mutant virus VABV600 was further typed with a panel ofN-specific MAbs (Table 2). Not only the binding of D-specific MAbSDOW17, but also the binding of D-specific MAbs 130.2, 130.4, 131.7, and131.9 and WBE1 to vABV600 was greatly reduced. If hybridoma culturesupernatant of these MAbs was diluted to 0.3-0.5 μg IgG/ml brightstaining was observed for wild type PRRSV, but no staining could beobserved for vABV600. However, when the IgG of MAbs 130.2, 130.4, 131.7,and 131.9 was purified and used more concentrated (10 μg IgG/ml) faintstaining was observed. Staining of vABV600 with A- and B-specific MAbswas comparable to PRRSV. These data indicated that we have created avirus that is antigenically different from wild type PRRSV or NorthAmerican PRRS viruses.

[0071] 3: Identification of Replication Signals at the 3′ End of thePRRSV Genome

[0072] In order to determine cis-acting sequences that are essentialsignals for RNA replication (plus and/or minus strand synthesis and/orsubgenomic mRNA synthesis), several deletions were made in theinfectious cDNA clone and transcripts derived from these deletionmutants were analysed for replication in BHK-21 cells. When transcriptsfrom pABVS21, lacking the entire ORF7 gene were transfected to BHK-21cells, the expression of the N-protein could not be detected in IPMA(FIG. 2). Interestingly, these transcripts were also defective in theexpression of other structural proteins, such as GP₃, GP₄ and M. Thisindicated that these RNAs were not replicated and did not producesubgenomic RNAs. On the other hand, the deletion of ORF2, ORF3, ORF4,ORF5 and the 5′ end ORF6 from the infectious copy (pABV594) resulted inviral RNA that was still capable of replication. Therefore, replicationsignals are present in the coding region of ORF7 and not in the codingregion of ORF's 2-6. To test this and further locate the regionsinvolved in replication, mutants containing smaller deletions in ORF7were constructed. The transcripts of these constructs were tested fortheir ability to replicate by detecting the expression of PRRSV proteinsin IPMA of transfected BHK-cells (FIG. 2). From these results, it couldbe concluded that essential signals for replication of the PRRSV genomeare present between nucleotides 14643 to 14687. Viral RNAs lacking thisregion were defective in replication.

[0073] 4. Analysis of Full Length cDNA Clone pABV575 Lacking the NdeISite in ORF6

[0074] A full length cDNA clone, pABV575, was created that had a uniqueNdeI site at position 12559 due to mutation of the second NdeI site atposition 14265 by PCR. RNA was produced from pABV575 and transfected toBHK-21 cells together with RNA from its parent clone pABV437. At 24 hafter transfection with pABV575 RNA and pABV437 RNA an equal number ofcells stained positive in IPMA with M-specific and N-specific MAbs (FIG.3). Furthermore, the intensity of the staining was similar. However,when the supernatant of the transfected BHK-21 cells was transferred toPAMs and incubated for 24 h, the number of cells infected by vABV575 wasmuch lower than that observed for vABV437. Furthermore, the cpedeveloped much slower in the PAMs inoculated with vABV575 than withvABV437. Although the replication of the RNA and synthesis of thesubgenomic RNAs of vABV575 in BHK-21 appeared to be at the wild typelevel, the virus that is produced was less infectious for macrophages.This was most likely due to the amino acid mutation in the M protein(Thr→Asn) that resulted from the destruction of the NdeI site atposition 14265.

[0075] 5: Introduction of an HA-Tag in the Infectious Clone of PRRSV

[0076] An epitope of the haemagglutinin of influenza A (HA-tag;Kolodziej et al., 1991) was expressed by different recombinant PRRSVviruses. The HA epitope was chosen as foreign antigen for expression inPRRSV mainly for two reasons; First, the tag has a limited size (27nucleotides), which reduces the chance to disturb the replication of thevirus or the expression or function of the protein to which it is fused.Second, antibodies to detect the expression of this epitope areavailable. The HA-tag was introduced at the 5′ end of ORF7 (pABV525),and at the 3′ end of ORF7 (pABV526; FIG. 4) as such that it did notinduce mutations in other ORFs. We expected to get high expression ofthe foreign antigen by inserting it in the ORF7 gene, because subgenomicmessenger RNA7(encoding ORF7) is most abundantly produced in infectedcells. Since we could not predict the influence of the HA-epitope on thefunction and the structure of the N protein, we created an additional inframe insertion of the 16-amino acid self-cleaving 2A protease offoot-and-mouth disease virus (FMDV; Percy et al., 1994). This proteasewas introduced downstream of the HA-tag at the 5′ end of ORF7, whichresulted in clone pABV523 (FIG. 4). We expected that this would resultin the expression of a polyprotein, which could be proteolyticallycleaved to release both the HA-tag and the N-protein.

[0077] 5. Analysis of Recombinants of PRRSV Expressing the HA Epitope

[0078] First, the expression of the structural proteins by the varioustranscripts from the recombinant full-length cDNA clones was tested inIPMA. BHK-21 cells, transfected with transcripts of pABV523, 525, and526 stained positive with MAbs directed against GP₃, GP₄, the M protein,and the N protein, which indicated that these PRRSV proteins wereproperly expressed (FIG. 4). The cells also stained positive with a MAbdirected against HA, indicating that the HA epitope was expressed by allthree RNAs. Therefore, the HA-expressing transcripts replicated inBHK-21 cells. In addition, the N-protein to which the HA-tag was fusedwas still expressed by the mutant RNAs.

[0079] To examine whether the transcripts of pABV523, 525 and 526 wereable to produce infectious virus, the culture supernatant of transfectedBHK-21 cells was used to infect PAMs.

[0080] PAMs not only stained positive with MAbs directed against thePRRSV proteins GP₃, GP₄, M protein and N protein in IPMA, but also withMAb 12CA5 directed against the HA epitope. However, when PAMs weredouble stained, both with MAbs against the HA-tag and the N protein, wealso detected PAMs which could only be stained with the MAb against theN protein but not with that against the HA-tag. For viruses derived frompABV525 and pABV526 the percentage of cells that stained only withN-specific Mabs was higher than for the viruses derived form pABV523,which contained the additional protease 2A. This indicated that theHA-tag directly attached to the N- or C-terminus of the N proteindisturbed to some extent either the packaging of the viral RNA or theinfectivity of the virus. However, when the protease 2A was introducedto cleave the HA-tag from the N protein by the protease 2A, the fitnessof the resulting virus (vABV523) was not or hardly reduced (FIG. 4). Therecombinant viruses were designated vABV523, vABV525 and vABV526.

[0081] Analysis of protease 2A activity in vABV523 The activity of theprotease 2A was further analyzed by radioimmunoprecipitation. Besides a15 kDa protein, an additional protein of approximately 18 kDa wasimmunoprecipitated with N-specific MAb 122.17 from cells transfectedwith transcripts of pABV523. The 15 kDa protein was similar in size tothe wild type N protein; the 18 kDa protein resembled the expected sizeof the polyprotein of HA-protease 2A-N. These data indicated thatprotease 2A of FMDV is able to cleave the HA-protease 2A-N polyproteinin the cell, which results in the release of the HA-tag from the Nprotein.

[0082] 5.Growth Characteristics of HA-Expressing Viruses

[0083] The amount of virus produced by BKH-21 cells transfected withtranscripts from pABV437 and pABV523 was generally higher than thatproduced by BHK-21 cells transfected with transcripts from pABV525 andpABV526.

[0084] Serial passage of HA-expressing viruses on PAMs resulted instocks of vABV523, vABV525, and vABV526 with titers of approximately 10⁷TCID₅₀/ml. It needs to be resolved whether the HA-expressing viruseshave the same growth properties as the wild type virus of the infectiouscopy of PRRSV (vABV437). This will be studied in growth curves.

[0085] 5. Analysis of the Stability of HA-Expressing Viruses.

[0086] To determine the stability of HA-expressing viruses, viral RNAwas examined at passage 4. For this purpose, RT-PCR was performed onisolated viral RNA. Part of the ORF7 gene, the site at which the HA-tagwas inserted, was amplified by PCR and the obtained fragments wereanalyzed on agarose gel. We obtained two fragments for vABV523 andvABV525 and one fragment for vABV526. Sequence analysis of the mostabundantly amplified fragment showed that vABV523 at passage 4 stillcontained the properly inserted nucleotide sequence encoding the HA-tagand the protease 2A gene. In contrast, both vABV525 and vABV526 had lostthe inserted nucleotide sequence encoding the HA-tag.

[0087] 1. Mutation of Cys-27 and Cys-76 in the N Protein Inhibits theProduction of Infectious Particles of PRRSV

[0088] In this study we have found that mutation of Cys-27→Asn andCys-76→Leu in the N protein of PRRSV interferes with the production ofinfectious particles in BHK-21 cells. We conclude that these residuesare essential for a proper structure or function or both of the Nprotein in virus assembly of PRRSV. The N protein is involved in thefirst steps in virus assembly, the binding of the viral genomic RNA andformation of the capsid structure. Since transcripts of genomic lengthcDNA clones containing the Cys-27→Asn and Cys-76→Leu replicated at thewild type level, the mutations in the Cys residues destroy the bindingof the RNA by the N protein. Alternatively, they induce a differentstructure of the N protein that inhibits the formation of propercapsids. The defect in the encapsidation of the viral RNA genome can becomplemented by wild type N protein transiently expressed orcontinuously expressed in a (BHK-21) cell line. In this way a virus isproduced that is able to complete only one round ofinfection/replication. Therefore such a virus is considered to be a verysafe vaccine for protection against PRRSV in pigs.

[0089] 2. Introduction of a Marker in the N Protein of PRRSV.

[0090] The aim of this study was to create mutant PRRS viruses that canbe serologically differentiated from field virus and therefore may bepromising mutants for marker vaccine development against PRRSV. The Nprotein was chosen as a first candidate for mutagenesis to create avirus with a serologic marker since many studies have shown that the Nprotein is the most antigenic protein of PRRSV. For example, pigsinfected with PRRSV develop strong antibody responses against the Nprotein of PRRSV (Meulenberg et al., 1995). In addition, the N proteincontains two antigenic regions designated B and D that are conserved inEuropean and US PRRSV isolates and MAbs directed to these regions areavailable (Meulenberg. et al., 1998). Here, we have demonstrated thatmutation of 4 amino acids in site B to corresponding amino acids of theEAV N protein and mutation of 5 or 9 amino acids in domain D tocorresponding amino acids of the LDV N protein inhibited the productionof infectious virus particles. Since RNA replication and subgenomic mRNAsynthesis appeared to be at the wild type level, these mutations mostlikely prevented the formation of proper capsids by the N protein.However, mutagenesis of a single amino acid in the D region (Asp-62→Tyr)resulted in virus vABV600 that had a different MAb binding profile fromPRRSV and all other PRRSV Viruses. vABV600 induces a different spectrumof antibodies in pigs, compared to these other PRRSV isolates. ThereforevABV600 can be differentiated from field virus on the basis of serumantibodies and is an excellent mutant for further development of markervaccines against PRRSV.

[0091] 3: Elucidation of Replication Signals in ORF7 of Lelystad Virus

[0092] It has been shown for many positive strand RNA viruses that their5′ and/or 3′ noncoding regions contain essential signals that controlthe initiation of plus- and minus-strand RNA synthesis. It was not yetdetermined for PRRSV whether these sequences alone are sufficient forreplication. The production of an infectious clone allowed us to analysereplication signals in the genome of PRRSV. In this study we have mappedcis-acting sequence elements required for replication by introducingdeletions in the infectious clone. We have shown that transcriptsderived from cDNA clones lacking the ORF7 gene are not replicated. Amore systematic deletion analysis showed that a region of 44 nucleotidesbetween nucleotides 14644 to 14687 in the ORF7 gene was important forreplication of RNA of PRRSV. This was an essential interesting finding,since the sequences essential for replication of most positive strandRNA viruses are present in the 5′ and 3′ noncoding regions. It is alsoan important finding for studies who's aim is to develop viral repliconswhich can only be rescued in complementing cell lines expressing thedeleted ORFs. The minimal sequence requirements for these RNAs are 5′noncoding region-ORF1a-ORF1b-ORF7-3′ noncoding region. Viral RNA s orreplicons containing these sequence elements supplemented with aselection of fragments from other PRRSV open reading frames or fragmentsof open reading frames expressing antigens of other (heterologous)pathogens can be packaged into virus particles when the proteinsessential for virus assembly are supplied in trans. When these particlesare given to pigs, for example as vaccine, they will enter specific hostcells such as macrophages and virus- or heterologous antigens areexpressed and induce immune responses because of the replicating RNA.However, since the RNA does not express (all) the proteins required forpackaging and the production of new particles, the replicon can notspread further, creating an extremely efficient, but safe andnot-spreading recombinant vaccine effective against PRRSV and/orheterologous pathogens.

[0093] 4. Production of an Attenuated PRRSV Virus by Deletion of theNdeI Site in ORF6.

[0094] In this study we have produced a mutant PRRS virus vABV575 thathad different growth characteristics in PAMs compared to the parentstrain vABV437. Whereas no difference in the expression of structuralproteins in BHK-21 cells by RNAs of vABV575 or vABV437 was observed, thevABV575 virus produced in BHK-21 cells infected PAMs slower thanvABV437. The growth kinetics of vABV575 need to be analyzed further byperforming growth curves in PAMs. In the cDNA clone pABV575, that wasused to produce vABV575, the NdeI site at position 14265 in ORF6 wasmutated. This resulted in an amino acid change of Thr-59->Asn in the Mprotein. The mutated M protein was still bound by M-specific MAb 126.3.The M protein is the most conserved structural protein amongarteriviruses and coronaviruses. The protein is an integral membraneprotein containing three N-terminal hydrophobic membrane spanningdomains (Rottier, 1995). The protein spans the membrane three timesleaving a short N-terminal domain outside the virion and a shortC-terminal domain inside the virion. The M protein of coronaviruses wasshown to play an important role in virus assembly (Vennema et al.,1996). The Thr-59→Asn mutation is located between the second and thirdmembrane spanning fragment of M in AB575. This mutation influences virusassembly, the stability of the virus, or virus entry in the PAMs.

[0095] 5. Expression of the HA Epitope in Recombinant PRRSV viruses

[0096] In this study we have successfully used PRRSV as a vector for theexpression of a foreign antigen, an HA epitope of the haemagglutinin ofinfluenza A virus. Recombinant PRRSV viruses were engineered thatproduced the HA tag fused to the N- or C- terminus of the N protein. Inaddition, a PRRSV mutant was created that contained the HA-tag as wellas the protease 2A of FMDV fused to the N terminus of the N protein. Theprotease 2A was functionally active in the context of the PRRSV virus,and cleaved the HA-tag from the N protein. This resulted in an N proteinthat is identical to the wild type N protein, except for the first andsecond amino acids (Met and Ala) that are lacking in the mutant. Geneticanalysis of passage 4 of the recombinant viruses indicated that themutant virus containing both the HA-tag and the protease 2A was morestable than the mutant viruses expressing the HA-N-fusion proteins.Apparently, the lack of the first methionine? and mutation of the secondamino acid at the N-terminus of N is better tolerated by the virus thanthe addition of the HA-tag of 9 amino acids to the N- or C-terminus ofN. Further genetic and functional analysis needs to be done to explainthe differences in stability observed for these viruses. In addition,pigs need to be infected with these HA-expressing mutants to determinewhether antibody responses are induced against the HA epitope.

[0097] The ORF7 gene was selected for insertion of the HA-tag mainly fortwo reasons; (I) The subgenomic RNA7 expressing this gene is the mostabundant subgenomic RNA produced in infected cells and (II) the HA-tagcould be inserted without mutating other ORFs since ORF7 has very littleoverlap with ORF6 at the 5′ end and no overlap with other ORFs at the 3′end. However, similar constructs can be made by introducing the HA-tagand protease 2A at the 5′ end of ORP2 and at the 5′ end of ORF5 withoutaffecting other ORFs.

[0098] The successful expression of the HA-tag in combination with theprotease 2A at the 5′ end of ORF7 creates new opportunities to expressother foreign antigens such as the E2 protein of hog cholera virus, or Bcell epitopes of parvo virus by PRRSV. Since PRRSV specifically infectsmacrophages, cells of the immune system that have antigen presentationand processing capacities, PRRSV might be an excellent vector for theexpression of antigens and induction of immunity to these antigens inthe pig.

LEGENDS TO THE FIGURES

[0099]FIG. 1. Properties of full length cDNA clones of PRRSV containingmutations in the ORF7 gene. The mutated ORF7 genes were inserted ininfectious cDNA clone pABV437 with the unique HpaI and PacI site thatare indicated. The plasmid (pABV) numbers of the resulting constructsare shown. RNA replication was determined by detecting the expression ofstructural proteins in IPMA after transfection of the transcripts of thefull length cDNA clones in BHK-21 cells. N protein production wasdetermined in IPMA or immunoprecipitation. Production of infectiousvirus was established by transfer of the supernatant of transfectedBHK-21 cells to PAMs and detection of cpe.

[0100]FIG. 2. Properties of full length cDNA clones of PRRSV containingdeletions in the region encoding the structural proteins of LV in orderto elucidate the presence of replication signals in this region. Thedeleted regions (dotted bars), the regions of ORF7 still present (darkbars) and the plasmid (pABV) numbers of the resulting clones are shown.RNA replication was determined by detecting the expression of structuralproteins, and the expression of the N-protein in particular, both inIPMA. Production of infectious virus was established by infecting PAMswith the supernatant of transfected BHK-21 cells. IPMA was performed todetect the expression of LV-proteins.

[0101]FIG. 3. Properties of infectious cDNA clone pABV575. This clonewas constructed by mutation of the NdeI site at position 14265 in ORF6.RNA replication was determined by detecting the expression of structuralproteins in IPMA after transfection of the transcripts of the fulllength cDNA clones in BHK-21 cells. Production of infectious virus wasestablished by transfer of the supernatant of transfected BHK-21 cellsto PAMs and detection of cpe.

[0102]FIG. 4. Introduction of an antigenic marker in the infectiousclone of PRRSV. The insertion of the HA tag and protease 2A sequence inplasmids pABV 525, 523 and 526 is indicated. RNA replication wasdetermined by detecting the expression of structural proteins in IPMAafter transfection of the transcripts of the full length cDNA clones.The expression of N and HA was also determined in IPMA. Production ofinfectious virus was established by transfer of the supernatant oftransfected BHK-21 cells to PAMS and detection of cpe.

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[0110] 8. Murtaugh, M. P., Elam, M. R., and Kakach, L. T., (1995).Comparison of the structural protein coding sequences of the VR-2332 andLelystad virus strains of the PRRS virus. Arch. Virol. 140, 1451-1460.

[0111] 9. Percy, N., Barclay, W. S., Garcia-Sastre, A. and Palese, P.Expression of a foreign protein by influenza A virus. 1994. J. Virol.68: 4486-4492

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[0113] 11. Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecularcloning a laboratory manual. 1989. 2nd edition, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.

[0114] 12. van Nieuwstadt, A. P., Meulenberg, J. J. M., vanEssen-Zandbergen, A., Petersen-den Besten, A., Bende, R. J., Moormann,R. J. M., and Wensvoort, G. (1996). Proteins encoded by ORFs 3 and 4 ofthe genome of Lelystad virus (Arteriviridae) are structural proteins ofthe virion. J. Virol. 70, 4767-4772.

[0115] 13. Wensvoort, G., Terpstra, C., Pol, J. M. A., Ter Laak, E. A.,Bloemraad, M., de Kluyver, E. P., Kragten, C., van Buiten, L., denBesten, A., Wagenaar, F., Broekhuijsen, J. M., Moonen, P. L. J. M.,Zetstra, T., de Boer, E. A., Tibben, H. J., de Jong, M. F., van't Veld,P., Groenland, G. J. R., van Gennep, J. A., Voets, M. Th., Verheijden,J. H. M., and Braamskamp, J. (1991). Mystery swine disease in theNetherlands: the isolation of Lelystad virus. Vet. Quart. 13, 121-130.

[0116] 14. Vennema, H., Godeke, G. -J., Rossen, J. W. A., Voorhout, W.F., Horzinek, M. C., Opstelten, D. -J. E., and Rottier, P. J. M. (1996)Nucleocapsid-independent assembly of coronavirus-like patricales byviral envelope protein genes. EMBO J. 15, 2020-2028

[0117] Wensvoort, G., Terpstra, C., Boonstra, J., Bloemraad, M., and vanZaane, D. (1986). Production of monoclonal antibodies against swinefever virus and their use in laboratory diagnosis. Vet. Microbiol. 12,101-108. TABLE 1 Primers used in PCR-mutagenesis and sequencing Sense(+) Primer (nt.) Sequence primer^(a) antisense(-) Purpose LV975′CATTGCACCCAGCAACGGTTCAGTTGT 3′ − Cys-27→Asn LV1005′CGTCTGGATCGATTGCAAGAGGAGGGA 3′ − Cys-76→Leu LV1885′TCTGGATCGATTGCAAGCAGAGGGAGCGTTCAGTCTGGG − Asp-62→TyrTGAGGTGGTGCCGGATGTCATATTCAGCAG 3′ LV27 5′GATTGGATCCAACACATCATTCGAGCTG3′ + Δ Ndel LV182 5′GGATTGAAAATGCAATTAATTCATGTAT 3′ − Δ Ndel 118U250(14755) 5′CAGCCAGGGGAAAATGTGGC 3′ − Sequencing LV37 (14340)5′GATTGGATCCACCATGGAGTCATGGAAGTTTATCACT 3′ + Sequencing LV75 (15088)5′TCTAGGAATTCTAGACGATCG 3′ − Sequencing LV76 (15088)5′TCTAGGAATTCTAGACGATCG(T)₄₀3′ − RT-PCR LV82 (14703)5′AGCAACCTAGGGGAGGACAG 3′ + Sequencing LV108 (14566)5′GGAGTGGTTAACCTCGTCAAGTATGGCCGGTAAAAACCAGAGCC 3′ + ORF7-HA LV112(14958) 5′CCATTCACCTGACTGTTTAATTAACTTGCACCCTGA 3′ − Pacl site LV139(14609) 5′AACTTTGACCTTCTCAAGTTGGCCGGCGACGTCGAGTCCAA + 1^(st)HA-prot-ORF7 CCCAGGGCCCGGTAAAAACCAGAGCCAGAA 3′ LV140 (14609)5′GAGTGGTTAACCTCGTCAAGTATGGCCGGTAAATACCCATACGAT + 2^(nd) HA-prot-ORF7GTTCCAGATTACGCT0AACTTTGACCTTCT 3′ LV188 (14687)5′ACGTGCGTTAACTAAGGTGCAATGATAAAGTCCCA 3′ + Δ 99 nt. 5′ORF7 LV189 (14796)5′ACGTGCGTTAACTAAATCCGGCACCACCTCACCCA 3′ + Δ 198 nt. 5′ORF7 LV190(14885) 5′ACGTGCGTTAACTAAGGGAAGGTCAGTTTTCAGGT 3′ + Δ 297 nt. 5′ORF7LV191 (14936) 5′ACGTGCGTTAACTAACGCCTGATTCGCGTGACTTC 3′ + Δ 348 nt.5′ORF7 LV192 (14609) 5′AAATACCCATACGATGTTCCAGATTACGCTAACCAGAGCCA 3′ +1^(st) HA-ORF7 LV193 (14609) 5′AGTGGTTAACCTCGTCAAGTATGGCCGGTAAATACCCATACG 3′ = 2^(nd) HA-ORF7 LV194 (14971)5′ACTGTTTAATTAAGCGTAATCTGGAACATCGTA − ORF7-HA TGGGTAACTTGCACCCTG 3′LV195 (14642) 5′ACGTGCGTTAACTAACCGATGGGGAATGGCCAG 3′ + Δ 55 nt 5′ORF7LV196 (14642) 5′GGAGTGGTTAACCTCGTCAAGTAACCGATGGGGAATGGCCAG 3′ + Δ 45 nt5′ORF7 LV197 (14597) 5′ACGTGCGTTAACGGCCGGTAAAAACCAGAGC 3′ + Δ 10 nt3′ORF6 LV198 (141333) 5′GCTCGTGCTAGCCTTTAGCATCACATACAC 3′ + Δ 54 nt3′ORF6 LV199 (14596) 5′CTTGACGAGGTTAACTGGTACTAGAGTGCC 3′ − Δ 54 nt3′ORF6

[0118] TABLE 2 Staining of LV4.2.1, vABV600 (Asp-62→Tyr mutation),ATCCVR2332-like PRRSV containing an Asp61→ Tyr mutation in the Nprotein, and ATCC-VR2332 with various N-specific MAbs in IPMA vABV600ATCCVR2332- (Asp-62→ like (Asp61→ MAb Site LV4.2.1 Tyr) Tyr) ATCC-VR2332138.22 A + + − − NS99 B + + + + 122.17 D ++ ++ ++ ++ 130.2 D ++ −¹⁾ − +130.4 D ++ −¹⁾ − + 131.7 D ++ −¹⁾ − + 131.9 D ++ −¹⁾ − + SDOW17 D ++ −¹⁾−¹⁾ ++ WBE1 D + − − − WBE4 D ++ ++ − − WBE5 D ++ ++ − − WBE6 D ++ ++ − −VO17 ? − − + +

[0119]

1 29 1 6 PRT Porcine reproductive and respiratory syndrome virus 1 GlnLeu Cys Gln Leu Leu 1 5 2 17 PRT Porcine reproductive and respiratoryvirus 2 Pro Glu Lys Pro His Phe Pro Leu Ala Ala Glu Asp Asp Ile Arg His1 5 10 15 His 3 11 PRT Porcine reproductive and respiratory virus 3 IleSer Thr Ala Phe Asn Gln Gly Ala Gly Thr 1 5 10 4 26 DNA ArtificialSequence PCR Primer 4 cattgcaccc agaactggtt cagttg 26 5 27 DNAArtificial Sequence PCR Primer 5 cgtctggatc gattgcaaga ggaggga 27 6 69DNA Artificial Sequence PCR Primer 6 tctggatcga ttgcaagcag agggagcgttcagtctgggt gaggtggtgc cggatgtcat 60 attcagcag 69 7 28 DNA ArtificialSequence PCR Primer 7 gattggatcc aacacatcat tcgagctg 28 8 28 DNAArtificial Sequence PCR Primer 8 ggattgaaaa tgcaattaat tcatgtat 28 9 20DNA Artificial Sequence PCR Primer 9 cagccagggg aaaatgtggc 20 10 37 DNAArtificial Sequence PCR Primer 10 gattggatcc accatggagt catggaagtttatcact 37 11 21 DNA Artificial Sequence PCR Primer 11 tctaggaattctagacgatc g 21 12 22 DNA Artificial Sequence PCR Primer 12 tctaggaattctagacgatc gt 22 13 20 DNA Artificial Sequence PCR Primer 13 agcaacctaggggaggacag 20 14 44 DNA Artificial Sequence PCR Primer 14 ggagtggttaacctcgtcaa gtatggccgg taaaaaccag agcc 44 15 36 DNA Artificial SequencePCR Primer 15 ccattcacct gactgtttaa ttaacttgca ccctga 36 16 72 DNAArtificial Sequence PCR Primer 16 aactttgacc ttctcaagtt ggccggcgacgtcgagtcca acccagggcc cggtaaaaac 60 cagagccaga ag 72 17 75 DNAArtificial Sequence PCR Primer 17 gagtggttaa cctcgtcaag tatggccggtaaatacccat acgatgttcc agattacgct 60 aactttgacc ttctc 75 18 35 DNAArtificial Sequence PCR Primer 18 acgtgcgtta actaaggtgc aatgataaag tccca35 19 35 DNA Artificial Sequence PCR Primer 19 acgtgcgtta actaaatccggcaccacctc accca 35 20 35 DNA Artificial Sequence PCR Primer 20acgtgcgtta actaagggaa ggtcagtttt caggt 35 21 35 DNA Artificial SequencePCR Primer 21 acgtgcgtta actaacgcct gattcgcgtg acttc 35 22 41 DNAArtificial Sequence PCR Primer 22 aaatacccat acgatgttcc agattacgctaaccagagcc a 41 23 42 DNA Artificial Sequence PCR Primer 23 agtggttaacctcgtcaagt atggccggta aatacccata cg 42 24 51 DNA Artificial Sequence PCRPrimer 24 actgtttaat taagcgtaat ctggaacatc gtatgggtaa cttgcaccct g 51 2533 DNA Artificial Sequence PCR Primer 25 acgtgcgtta actaaccgatggggaatggc cag 33 26 42 DNA Artificial Sequence PCR Primer 26 ggagtggttaacctcgtcaa gtaaccgatg gggaatggcc ag 42 27 31 DNA Artificial Sequence PCRPrimer 27 acgtgcgtta acggccggta aaaaccagag c 31 28 30 DNA ArtificialSequence PCR Primer 28 gctcgtgcta gcctttagca tcacatacac 30 29 30 DNAArtificial Sequence PCR Primer 29 cttgacgagg ttaactggta ctagagtgcc 30

1. A porcine reproductive and respiratory virus (PRRSV) replicon havingat least some of its original PRRSV nucleic acid deleted, said repliconcomprising essential elements from the PRRSV polymerase region andcapable of in vivo RNA replication.
 2. A porcine reproductive andrespiratory syndrome virus (PRRSV) replicon capable of in vivo RNAreplication comprising PRRSV nucleic acid and nucleic acid derived fromat least one heterologous microorganism.
 3. The PRRSV replicon of claim1, further comprising nucleic acid derived from at least oneheterologous microorganism.
 4. The PRRSV replicon of any one of claims 1to 3, wherein said replicon comprises RNA.
 5. The PRRSV replicon of anyone of claims 1 to 4, wherein said essential elements comprise essentialnucleic acid sequence elements from the 5′ noncoding region, the ORF1a,the ORF 1b, the ORF7 and the 3′ noncoding region of PRRSV.
 6. The PRRSVreplicon of any one of claims 1 to 5, said essential elements compriseat least a nucleic acid corresponding to a 44 nucleotide region betweennucleotides 14643 to 14687 in the ORF 7 region of PRRSV, as identifiedin the nucleic acid sequence of the Lelystad virus isolate.
 7. The PRRSVreplicon of any one of claims 1 to 6, further comprising at least onenucleic acid modification in the ORF7 gene encoding the N proteinrendering it incapable of N-protein capsid formation during virusassembly.
 8. The PRRSV replicon of claim 7, wherein said nucleic acidmodification comprises a nucleic acid modification deleting a cysteinein the N-protein.
 9. The PRRSV replicon of claim 7, wherein said nucleicacid modification comprises a nucleic acid modification leading to anamino acid change of the B-region comprising amino acids 25-30 of theN-protein (SEQ ID NO: 1).
 10. The PRRSV replicon of claim 7, whereinsaid nucleic acid modification comprises a nucleic acid modificationleading to an amino acid change of the D-region comprising amino acids51-67 of the N-protein (SEQ ID NO: 2).
 11. The PRRSV replicon of claim7, wherein said nucleic acid modification comprises a nucleic acidmodification leading to an amino acid change of the B-region comprisingamino acids 80-90 of the N-protein (SEQ ID NO: 3).
 12. The PRRSVreplicon of any one of claims 1 to 11, further comprising a markerallowing serological discrimination.
 13. The PRRSV replicon of claim 12,wherein said marker comprises a nucleic acid modification leading to anamino acid change of the original asparagine residue at position 62 ofthe N-protein.
 14. The PRRSV replicon of claim 12, wherein said markercomprises a nucleic acid modification leading to an amino acid change inone or more ORFs selected from the group comprising ORF 2,3,4,5 and 6.15. The PRRSV replicon of any one of claims 1 to 14, further comprisinga nucleic acid modification in a virulence marker of PRRSV.
 16. ThePRRSV replicon of claim 15, wherein said nucleic acid modificationcomprises a modification in ORF 6 encoding the virion membrane spanningM-protein.
 17. The PRRSV replicon of claim 16, wherein said nucleic acidmodification modifies protein M in between its second and third virionmembrane spanning fragment.
 18. The PRRSV replicon of any one of claims2 to 17, wherein said heterologous microorganism comprises amicroorganism that is pathogenic to pigs.
 19. The PRRSV replicon ofclaim 18, wherein said microorganism is a virus.
 20. A vaccinecomprising the PRRSV replicon of any one of claims 1 to
 19. 21. A methodof vaccinating pigs comprising: administering to a pig, the vaccine ofclaim 20 in an amount sufficient to produce immunity.